This invention relates generally to constructs and their use in gene expression or gene regulation assays. More particularly, the present invention provides expression vectors and/or reporter vectors providing kinetics of protein expression with improved temporal correlation to promoter activity. The present invention provides, inter alia, novel vectors and cell lines useful for modulating gene expression, identifying and analysing regulatory sequences, new targets and reagents for therapeutic intervention in human diseases and for drug-screening.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. A particularly important area of research is the use of expression vectors to study gene expression. However, until now, a real-time analysis of gene expression has been limited by the lack of suitably designed vectors.
Reporter assays permit an understanding of what controls the expression of a gene of interest e.g., DNA sequences, transcription factors, RNA sequences, RNA-binding proteins, signal transduction pathways and specific stimuli.
Furthermore, reporter assays can be used to identify aspects of gene regulation that serve as new targets for therapeutic intervention in human disease. Reporter assays can potentially be used to screen drugs for their ability to modify gene expression. However, the cost and time required for current reporter assay systems, together with the inaccuracies caused by the lengthy response times, has limited this application.
Genomic sequences have promoter sequences, generally upstream of the coding region, which dictate the cell specificity and inducibility of transcription and thereby affect the level of expression of protein products.
Specific sequence elements, typically rich in the nucleotide bases A and U and often located in the 3′-UTR of a gene, affect the stability of the mRNA and thereby affect the level of expression of the protein product. RNA-binding proteins bind certain mRNA sequences and thereby regulate mRNA stability and protein expression. Other sequences and trans-acting proteins modulate other post-transcriptional pathways such as translational efficiency, mRNA splicing and mRNA export into the cytoplasm.
A common application of gene reporter assays is the study of DNA sequences that regulate transcription. Typically, these sequences are located in the promoter region, 5′ of the transcription start site. Such DNA elements are tested by cloning them into a similar site within a reporter plasmid, such that they drive and/or regulate transcription and therefore, expression of reporter protein. The reporter protein should be distinguishable from endogenous proteins and easily quantified. Various reporter proteins are used, the most common being luciferase, chloramphenicol transferase (CAT) and β galactosidase (β-gal).
The reporter protein is quantified in an appropriate assay and often expressed relative to the level of a control reporter driven by a ubiquitous promoter such as for example the promoter SV40. The control reporter must be distinguishable from the test reporter and is contained on a separate vector that is co-transfected with the test vector and used to control for transfection efficiency. Such assays are based on the premise that cells take up proportionally equal amounts of both vectors. Transient transfections of plasmid vectors are most commonly used.
The assays described above are used to identify a promoter region or the specific elements within a promoter. Alternatively, they are used to study the response to various stimuli of a promoter or regulatory element. In some applications, the reporter constructs, or the transfected cells, are placed into an organism to study promoter function in vivo.
Another application of these reporter assays is the study or measurement of signal transduction pathways upstream of a specific promoter. For example, a promoter dependent on mitogen activated protein kinase (MAPK) for transcription can be linked to a reporter construct and used to measure the level of MAPK activation (or MAPK-dependent transcription) in cells. This technique can be utilized with a variety of informative promoters or enhancers and can be applied to cells or living organisms such as transgenic mice. For example, a photon camera can be used to measure luciferase reporter activity in whole mice containing a luciferase reporter linked to a promoter of interest (Contag, et al, 1997).
Luciferase is the most commonly used reporter assay for in vitro systems. The Dual Luciferase assay (DLA; Promega, Madison, Wis., USA), is an improvement over other luciferase based systems in that both test and control reporter can essentially be measured in the same assay. As an example of current use, a typical DLA protocol is provided as follows:
The putative promoter element is cloned upstream of a firefly luciferase reporter gene such that it drives its expression. This plasmid is transiently transfected into a cell line, along with a control plasmid containing the Renilla luciferase gene driven by the SV40 promoter. ˜2–50% of cells take up plasmid and express the reporters for ˜3 days. The kinetics of expression involve an increase during the first ˜24 h as luciferase protein accumulates, followed by a decrease from ˜48 h as the number of plasmids maintained within the cells declines. 24–48 h after transfection, cells are harvested and lysed. Cell lysates are incubated with substrates specific to firefly luciferase and activity (light emission) is measured using a luminometer (96 well plate or individual samples). Additional substrates are then added, which inactivate firefly luciferase but allow Renilla luciferase to generate light. Renilla luciferase activity can then be measured.
The level of firefly luciferase activity is dependent, not only on promoter activity, but also on transfection efficiency. This varies greatly, depending on the amount of DNA, the quality of the DNA preparation and the condition of the cells. The co-transfected control plasmid (Renilla luciferase driven by the SV40 promoter) is used to correct for these variables, based on the premise that Renilla luciferase activity is proportional to the amount of firefly luciferase plasmid taken up by the cells. Data are expressed as firefly luciferase activity/Renilla luciferase activity.
The disadvantages of the Dual Luciferase assay are as follows:                (i) Reagents are expensive and perishable and must be freshly prepared.        (ii) Generally this assay involves the preparation of cell lysates, which is time consuming and adds inaccuracy. e.g., loss of cells during lysis, pipetting errors, residual buffer/medium altering volumes.        (iii) Each sample yields only one datum point being the total activity of the cell population. No information is gained concerning the percentage of cells that express the reporter, nor the amount of expression per cell.        (iv) The transfection control (Renilla) does not always correct for huge variation in transfection efficiencies because:                    (a) Certain DNA preparations transfect/express poorly (perhaps due to reduced proportion of supercoiled DNA), but do not cause a corresponding decrease in the amount of co-transfected control plasmid.            (b) There is evidence of cross-talk between the promoters of the two plasmids, such that control reporter activity is dependent on the construct with which it is co-transfected, e.g., expression of Renilla luciferase seems highest when co-transfected with a plasmid containing a strong promoter. Interference between promoters has also limited, if not prevented, the use of single plasmids expressing both test and control reporters.            (c) A common application of both transcriptional and post-transcriptional studies is to measure activation/suppression by various stimuli (e.g., PMA, EGF, hormones). Unfortunately, SV40, RSV, TK and probably many other ubiquitously expressed promoters are activated by a variety of stimuli. Since these promoters are used to drive expression of the transfection control reporter (Renilla), these reporters do not give a true reflection of transfection efficiency following such treatments. (Ibrahim et al. 2000).            (d) Differences in the half-lives of firefly vs Renilla luciferase proteins and perhaps mRNAs make the whole system very time-sensitive.            (e) Rapidly diminishing light emission, particularly for Renilla luciferase, require absolute precision in the timing of measurement.            (f) The relatively long half-lives of luciferase proteins and mRNAs effectively mask temporal changes in transcription (e.g., following various stimuli or treatments).                        
In existing post-transcriptional/mRNA stability reporter assays, candidate elements, thought to affect mRNA stability are cloned into the corresponding region of a reporter vector (e.g., firefly luciferase) driven by a constitutive promoter such as SV40 or RSV. Changes in expression relative to the empty vector (same vector without element of interest) are assumed to be the result of altered mRNA stability or translational efficiency. More complex assays are required to distinguish the two possibilities. As with the preliminary described transfection assays, a transfection control plasmid (e.g., Renilla luciferase driven by a constitutive promoter such as SV40 or RSV) is co-transfected to allow correction for transfection efficiency. These assays suffer from the following additional disadvantages:                (1) Existing vectors were not designed for post-transcriptional studies and have no means for switching off transcription.        (2) The purpose of these protocols is to study the post-transcriptional effects of candidate mRNA elements. However, these elements can also affect transcription of the reporter at the level of DNA. Furthermore, since the endogenous promoter of the gene of interest is not used, any transcriptional effects seen may have little physiological relevance.        
Other systems for studying mRNA stability exist but involve direct measurement of the mRNA rather than a protein reporter. Due to the labour-intensive nature of protocols for quantifying mRNA, such systems are far more time consuming.
One system, for example, utilizes the c-fos promoter, which responds to serum induction with a brief burst of transcription. Putative instability elements are cloned into the 3-UTR of a Beta Globin (BBB) construct, which expresses the very stable beta globin mRNA under the control of a serum-inducible (c-fos) promoter. Transfected cells (generally NIH 3T3 cells) are first serum starved and then exposed to medium containing serum. The brief nature of the transcriptional response allows the kinetics of reporter mRNA degradation to be followed in a time course. This assay suffers from the following disadvantages:                (i) Quantifying mRNA rather than reporter protein is very time consuming and is therefore not applicable to rapid screening.        (ii) Can only be used in cells that support serum inducibility of the c-fos promoter. For example, many tumour cell lines maintain c-fos promoter activity in the absence of serum.        (iii) In cells such as NIH 3T3 cells, which do have the desired serum response, serum deprivation causes a cell cycle block and subsequent addition of serum, releases the cells from this block in a synchronous manner. Therefore, mRNA stability can only be measured in specific stages of the cell cycle.        (iv) In addition to activating the c-fos promoter, serum activates a multitude of other pathways, which introduce unwanted variables and prevent the study of more specific stimuli.        
In another assay, cells are treated with drugs, such as Actinomycin D that inhibit transcription from all genes. The mRNA levels are measured in a time course to determine mRNA degradation rates. This system is used to study endogenous genes and suffers from the following disadvantages:                (i) Transcriptional inhibitors are extremely toxic at doses required such that mRNA stability is often being measured in stressed or dying cells.        (ii) Transcription inhibitors possess numerous unwanted activities including stabilization of certain mRNAs.        (iii) The process blocks transcription from all genes such that many signal transduction cascades are blocked, whereas others are activated. Therefore, results may not be physiologically relevant.        (iv) The technique is extremely labour intensive.        (v) The technique is highly variable within and between assays.        (vi) The technique is often not sensitive enough for transient transfection reporter assays, particularly in cells with low transfection efficiency.        
There is a need therefore to develop improved vectors and systems for conducting gene expression assays and in particular post-transcriptional assays as well as assays that permit a more real-time determination of changes in gene expression.